Polyhydroxyalkanoate synthase and gene encoding the same

ABSTRACT

The present invention provides a PHA (polyhydroxyalkanoate) synthase useful in a process for preparing a PHA, a gene encoding the enzyme, a recombinant vector comprising the gene, a transformant transformed by the vector, a process for producing a PHA synthase utilizing the transformant and a process for preparing a PHA utilizing the transformant. A transformant obtained by introducing a PHA synthase gene from  Pseudomonas putida  P91 strain into a host microorganism is cultured to produce a PHA synthase or PHA.

This application is a continuation-in-part of application Ser. No.09/820,721, filed Mar. 30, 2001 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a polyhydroxyalkanoate (hereinafter, referredto as a “PHA”) synthase, a gene encoding the synthase, a recombinantvector containing the gene, a transformant transformed by the vector, aprocess for producing the PHA synthase utilizing the transformant, and aprocess for preparing the PHA utilizing the transformant.

2. Related Background Art

There have been reported a number of microorganisms producingpoly-3-hydroxybutyric acid (PHB) or another PHA and storing it therein(“Biodegradable Plastic Handbook”, edited by Biodegradable PlasticResearch Society, NTS Co. Ltd., p. 178-197 1995). These polymers may be,as conventional plastics, used for producing a variety of products by,for example, melt processing. Since they are biodegradable, they have anadvantage that they can be completely degraded by microorganisms in thenatural environment, and they do not cause pollution due to remaining inthe natural environment like many conventional polymer compounds.Furthermore, they are excellently biocompatible, and thus are expectedto be used in applications such as a medical soft member.

It is known that a composition and a structure of such a PHA produced bya microorganism may considerably vary depending on the type of amicroorganism used for the production, a culture-medium composition andculturing conditions. Investigations have been, therefore, mainlyfocused on controlling such a composition or structure for the purposeof improving physical properties of a PHA.

For example, Japanese Patent Application Nos. 7-14352 and 8-19227 andJapanese Examined Publication No. 6-15604 describe that Alcaligeneseutropus H16 (ATCC No. 17699) and its variants may produce3-hydroxybutyric acid (3HB) and its copolymer with 3-hydroxyvaleric acid(3HV) with various composition ratios by changing a carbon source duringculturing.

Japanese Patent Publication No. 2642937 has disclosed that PHA in whicha monomer unit is 3-hydroxyalkanoate with 6 to 12 carbon atoms may beproduced by supplying a non-cyclic aliphatic hydrocarbon as a carbonsource to Pseudomonas oleovorans (ATCC No. 29347).

Japanese Patent Application Laid-Open No. 5-7492 discloses methods inwhich Methylobacterium sp., Paracoccus sp., Alcaligenes sp., andPseudomonas sp. are contacted with a primary alcohol with 3 to 7 carbonatoms to produce a copolymer of 3HB and 3HV.

Japanese Patent Application Laid-Open No. 5-93049 and No. 7-265065 havedisclosed that Aeromonas caviae is cultured using oleic acid or oliveoil as a carbon source to produce a two-component copolymer of 3HB and3-hydroxyhexanoic acid (3HHx).

Japanese Patent Application Laid-Open No. 9-191893 has disclosed thatComamonas acidovorans IF013852 is cultured using gluconic acid and1,4-butanediol as carbon sources to produce a polyester having 3HB and4-hydroxybutyric acid as monomer units.

Furthermore, it has been reported that certain microorganisms producePHAs having a variety of substituents such as groups derived from anunsaturated hydrocarbon, ester, allyl, cyano, groups derived from ahalogenated hydrocarbon and epoxide. Recently, there have been attemptsfor improving physical properties of a PHA produced by a microorganismusing such a procedure.

As an example of such a polymer, a PHA having a phenyl group in its sidechain has been developed. For example, Makromol Chem., 191, 1957-1965(1990); Macromolecules, 24, 5256-5260 (1991); and Chirality, 3, 492-494(1991) have described production of a PHA comprising3-hydroxy-5-phenylvaleric acid (3HPV) as a monomer unit by Pseudomonasoleovorans, where there has been observed variation in polymer physicalproperties probably due to the presence of 3HPV.

As described above, microorganism-produced PHAs with variouscombinations of composition and structure have been obtained by varyingfactors such as the type of a microorganism used, a culture mediumcomposition and culturing conditions. However, each microorganism or PHAsynthase has significantly different substrate specificity. Therefore,it has been difficult to produce PHAs comprising different monomer unitsextensively suitable to a variety of applications using knownmicroorganisms or PHA synthases alone.

SUMMARY OF THE INVENTION

A PHA having a substituent in its side chain as described above may beexpected to be a “functional polymer” having significantly usefulfunctions and properties owing to the properties of the introducedsubstituent. It is, therefore, extremely useful and important to preparea gene encoding a PHA synthase from a microorganism which can produceand store a very useful polymer having both such functionality andbiodegradability; prepare a recombinant vector comprising the gene, atransformant transformed by the vector; and develop a process forproducing a PHA synthase utilizing the transformant and a process forpreparing a PHA utilizing the transformant.

In view of usefulness of such a PHA synthase useful in PHA production,an object of the present invention is to provide a PHA synthase, a geneencoding the enzyme, a recombinant vector comprising the gene, atransformant transformed by the vector, a process for producing a PHAsynthase utilizing the transformant and a process for preparing a PHAutilizing the transformant.

For developing a PHA having a novel side-chain structure useful as, forexample, a device material or a medical material, the inventors havesearched a novel microorganism capable of producing and storing thedesired PHA. Additionally, the inventors have intensely investigated forpreparing a gene encoding a PHA synthase from such a microorganism, arecombinant vector containing the gene, a transformant transformed bythe vector and developing a process for producing a PHA synthaseutilizing the transformant and a process for preparing a PHA utilizingthe transformant.

The inventors have finally found a novel microorganism capable ofproducing and storing a novel PHA comprising3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) represented by formula(2) as a monomer unit from synthetic 5-(4-fluorophenyl)valeric acid(PPVA) represented by formula (1) as a starting material, and designateit as P91 strain.

The inventors have also found that P91 strain in capable of producingand storing a PHA comprising 3-hydroxy-4-phenoxy-n-butyric acid (3HPxB)represented by formula (4) as a monomer unit from 4-phenoxy-n-butyricacid (PxBA) represented by formula (3) as a starting material.

An example of a microorganism capable of producing and storing a PHAcomprising 3HPxB as a monomer unit is Pseudomonas oleovorans involved ina process described in Macromolecules. 29, 3432-3435, 1996. This processis considerably different from the process using PxBA as a substrate inP91 strain in that 8-phenoxyoctanoic acid (PxOA) is used as a substrate.In addition, for a PHA produced, the above reported process provides acopolymer consisting of three monomer units, i.e.,3-hydroxy-8-phenoxyoctanoic acid derived from the substrate PxOA,3-hydroxy-6-phenoxyhexanoic acid as a byproduct derived from ametabolite and 3HPxB. On the other hand, P91 strain can produce a PHAcomprising 3HPxB derived from PxBA as a sole phenoxy-containing monomerunit. In this respect, P91 strain is basically different from the abovereported strain.

There are no reports describing microbial production of a PHA comprising3HPxD as a monomer unit using PxBA as a substrate or 3HPxB as a solephenoxy-containing monomer unit.

Microbiological properties of P91 strain according to this invention areas follows.

<Microbiological Properties of P91 Strain>

(Morphologic Properties)

Cell shape and size: Bacilliform, 0.6 μm×1.5 μm

Cell polymorphism: No

Motility: Yes

Sporulation: No

Gram stainability: Negative

Colonization: Circular, smooth in the overallperiphery, low convex,smooth surface, gloss, cream color

(Physiological Properties)

Catalase: Positive

Oxidase: Positive

O/F test: oxidized form

Reduction of a nitrate: Negative

Indole formation: Negative

Acidification of dextrose: Negative

Arginine dihydrolase: Positive

Urease: Negative

Esculin hydrolysis: Negative

Gelatin hydrolysis: Negative

β-Galactosidase: Negative

Fluorochrome production on King's B agar: Positive

(Substrate Assimilation Ability)

Dextrose: Positive

L-Arabinose: Negative

D-Mannose: Negative

D-Mannitol: Negative

N-Acetyl-D-glucosamine: Negative

Maltose: Negative

Potassium gluconate: Positive

n-Capric acid: Positive

Adipic acid: Negative

dl-Malic acid: Positive

Sodium citrate: Positive

Phenyl acetate: Positive

From these microbiological properties, the inventors have attempted tocategorize P91 strain according to Bergey's Manual of SystematicBacteriology, Volume 1 (1984) and Bergey's Manual of DeterminativeBacteriology 9th ed. (1994) to determine that the strain belongs toPseudomonas putida. Thus, the strain was designated as Pseudomonasputida P91. The inventors have deposited Pseudomonas putida P91 toPatent Microorganism Depository Center in the National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry, under thedeposition number of FERM P-17409. P91 strain has been internationallydeposited on the basis of the Budapest Treaty, and its internationaldeposition number is “FERM BP-7373”.

The inventors have intensely conducted investigation for solving theabove problems and finally have succeeded cloning a gene for a PHAsynthase from P91 strain to achieve this invention.

Specifically, a PHA synthase of this invention is characterized in thatit has the amino acid sequence of SEQ ID NO.:1 or 3. A PHA synthaseaccording to the present invention may include a mutant PHA synthasewhere at least one mutation including deletion, substitution or additionof at least one amino acid is introduced as long as it does notdeteriorate PHA synthase activity exhibited by a protein comprisingthese amino acid sequences.

The present invention also encompasses a PHA synthase gene coding a PHAsynthase comprising the amino acid sequence of SEQ ID NO.:1 or 3.Examples of a sequence of such a gene include SEQ ID NO.:2 or 4.Furthermore, a mutant PHA synthase gene encoding the above mutant PHAsynthase obtained by mutation of the sequence of SEQ ID NOs.:2 and 4 isincluded in a PHA synthase gene according to this invention.

The present invention also encompasses a recombinant vector comprisingthe above PHA synthase gene and a transformant transformed by therecombinant vector. The present invention also encompasses a process forproducing a PHA synthase comprising the steps of culturing thetransformant and isolating the PHA synthase from a culture obtained, anda process for preparing a PHA comprising the steps of culturing thetransformant and isolating the PHA from a culture obtained.

The present invention provides a PHA synthase, a gene encoding the PHAsynthase, a recombinant vector comprising the gene and a transformanttransformed by the recombinant vector. The PHA synthase gene accordingto the present invention is useful for preparing a PHA having variousphysical properties because it encodes a PHA synthase using a monomerhaving a novel side-chain structure as a substrate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be more detailed. A PHA synthase gene of thepresent invention is isolated from Pseudomonas putida P91 strain. First,a chromosome DNA is obtained from a strain having a PHA synthase gene.The chromosome DNA may be isolated by a known technique.

For example, after P91 strain is cultured in a LB medium or an M9 mediumsupplemented with an appropriate carbon source, a chromosome DNA isprepared as described by, for example, Marmer et al. in Journal ofMolecular Biology, Vol. 3. p. 208 (1961). The chromosome DNA thusobtained is digested using an appropriate restriction enzyme (e.g.,Sau3AI) and a fragment with a proper length is ligated with a ligatablevector digested with a restriction enzyme (e.g., BamHI) to prepare agene library. Herein, a proper fragment length varies, e.g., about 4000to 25000 bps for a usual plasmid vector and about 15000 to 30000 bps fora cosmid or phage vector. A proper length of DNA fragment may becollected by a known method such as a method using a sucrose densitygradient or using an agarose gel described in Molecular Cloning, ColdSpring Harbor Laboratory (1982).

A vector is a phage vector or plasmid vector which can autonomouslyreplicate in the host microorganism. Examples of phage or cosmid vectorsinclude pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgT10,λgt11, Charon4A and Charon21A. Examples of plasmid vectors include pBR,pUC, pBluescriptII, pGEM, pTZ and pET groups. Various shuttle vectorsmay be used, e.g., vectors which may autonomously replicate in aplurality of host microorganisms such as E. coli and Pseudomonas sp.These vectors may be also digested with a proper restriction enzyme toprovide a desired fragment as described above.

A chromosome DNA fragment may be ligated with a vector fragment using aDNA ligase. For example, a ligation kit (Takara Shuzo Co., Ltd., etc.)may be used. Thus, for example, a chromosome DNA fragment may be ligatedwith a vector fragment to prepare a mixture of recombinant plasmidscomprising various DNA fragments (hereinafter, referred to as a “genelibrary”). In addition to a method using a proper length of chromosomeDNA fragment, a gene library may be prepared by a method that mRNAs areextracted from P91 strain, purified and used for preparation of a cDNAfragment using a reverse transcriptase as described in MolecularCloning, Cold Spring Harbor Laboratory, 1982. Alternatively, a genelibrary is transformed or transduced to E. coli, and then the genelibrary may be amplified to a large amount as described in MolecularCloning, Cold Spring Harbor Laboratory, 1982.

A recombinant vector may be introduced into a host microorganism by aknown method. For example, when using E. coli as a host microorganism, acalcium chloride method (Journal of Molecular Biology, Vol. 53, p. 159(1970)), a rubidium chloride method (Methods in Enzymology, Vol. 68, p.253 (1979)), electroporation (Current Protocols in Molecular Biology,Vol. 1, p. 184 (1994)) may be used. When using a cosmid vector or phagevector, transduction may be conducted using in vitro packaging (CurrentProtocols in Molecular Biology, Vol. 1, p. 571 (1994)). Alternatively, amethod involving conjugational transfer may be used.

Then, a probe is prepared for obtaining a DNA fragment comprising a PHAsynthase gene of P91 strain.

Some base sequences have been reported for PHA synthase genes; forexample. Peoples, O. P. and Sinskey, A. J., J. Biol. Chem. 264, 15293(1989); Huisman, G. W. et al., J. Biol. Chem., 266, 2191 (1991); Pieper,U. et al., FEMS Microbiol. Lett., 96, 73 (1992); Timm, A. andSteinbuchel, A., Eur. J. Biochem., 209, 15(1992); Matsusaki. H. et al.,J. Bacteriol., 180, 6459 (1998).

From these reported sequences, a region where a sequence is preserved toa higher degree is selected for designing an oligonucleotide. Such anoligonucleotide includes, but not limited to, a sequence described inTimm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992). Anoligonucleotide may be synthesized using, for example, Custom SynthesisService, Amersham-Pharmacia Biotech.

Then, the designed oligonucleotide as a primer is subject to polymerasechain reaction (hereinafter, referred to as “PCR”) using a chromosomeDNA in P91 strain as a template to partially amplify the PHA synthasegene. The PCR amplified fragment thus obtained is homologous to the PHAsynthase gene of P91 strain to about 100%, and may be expected to give ahigher S/N ratio as a probe during colony hybridization and may allowstringency control in hybridization to be facilitated. The above PCRamplified fragment is labeled with an appropriate reagent and used forcolony-hybridization of the above chromosome DNA library to select a PHAsynthase gene (Current Protocols in Molecular Biology, Vol. 1, p. 603(1994)). The PCR amplified fragment may be labeled using a commerciallyavailable kit such as AlkPhosDirect (Amersham-Pharmacia Biotech).

A gene fragment comprising a PHA synthase gene may be selected by, inaddition to the above method using a genotype, a method using aphenotype where PHA synthesis is directly evaluated. The presence of PHAsynthesis may be detected by, for example, staining with Sudan Black B(Archives of Biotechnology. VOl. 71, p. 283 (1970)) or determination ofPHA accumulation by phase contrast microscopy.

A plasmid may be collected from E. coli selected by any of the abovemethods using an alkali method (Current Protocols in Molecular Biology,Vol. 1, p. 161 (1994)) to obtain a DNA fragment comprising a PHAsynthase gene. The DNA fragment obtained may be sequenced by, forexample, Sanger's sequencing method (Molecular Cloning, Vol. 2, p. 133(1989). Alternatively, it may be conducted by a dye-primer method or adye-terminator method using an automatic sequencer such as DNA Sequencer377A (Perkin Elmer).

After determining all the sequences, hybridization may be conductedusing a DNA fragment prepared by an appropriate method, such as chemicalsynthesis, PCR using a chromosome DNA as a template, or digestion of aDNA fragment comprising the sequence with a restriction enzyme as aprobe, to provide a gene of this invention.

SEQ ID NOs.:2 and 4 show the sequences of PHA synthase gene of thisinvention while SEQ ID NOs.:1 and 3 show the amino acid sequences codedby the genes. As described above, there may be mutations for one orseveral amino acids such as deletion, substitution or addition as longas the polypeptides having these amino acid sequences retain PHAproducing activity. In addition to those having the sequence coding theamino acids of SEQ ID NOs.:1 and 3, the present invention may include adegenerated isomer coding the same polypeptide which has the same aminoacid sequence and is different only in a degeneration codon. Mutationsuch as deletion, substitution and addition may be introduced by, e.g.,a site mutation introduction technique (Current Protocols in MolecularBiology Vol. 1, p. 811 (1994)).

A transformed microorganism of this invention may be produced byintroducing a recombinant vector of the present invention into a hostsuitable to an expression vector used during preparing the recombinantvector. Examples of microorganisms which may be used as a host includevarious bacteria such as Esherichia sp., Pseudomonas sp., Ralstonia sp.,Alcaligenes sp., Comamonas sp., Burkholderia sp., Agrobacterium sp.,Plabobacterium sp., Vibrio sp., Enterobacter sp., Rhizobium sp.,Gluconobacter sp., Acinetobacter sp., Moraxella sp., Nitrosomonas sp.,Aeromonas sp., Paracoccus sp., Bacillus sp., Clostridium sp.,Lactobacillus sp., Coryncbacterium sp., Artbrobacter sp., Achromobactersp., Micrococcus sp., Mycobacterium sp., Streptococcus sp., Streptomycessp., Actinomyces sp., Norcadia sp. and Methylobacterium sp. Besides theabove bacteria, yeasts and molds such as Saccharomyces sp. and Candidasp. may be used as a host.

When using a microorganism belonging to Pseudomonas sp., e.g., abacterium such as E. coli as a host, it is preferable that therecombinant vector of the present invention itself can autonomouslyreplicate in a host used while comprising a constitution required forexpression such as a promoter, a DNA comprising a PHA synthase gene anda transcription termination sequence. Expression vectors include pLA2917(ATCC 37355) having a RK2 replication origin which may be replicated andretained by a range of hosts or pJRD215 (ATCC 37533) having a RSF1010replication origin, but any vector having a replication origin which maybe replicated and retained by a wide range of hosts may be used.

Any promoter which may be expressed in a host may be used; for example,promoters derived from E. coli, a phage, etc. such as trp. trc, tac,lac, PL, PR, T7 and T3 promoters. A recombinant DNA may be introduced ina bacterium by an appropriate procedure such as the above calciumchloride method and electroporation.

When using a yeast as a host, an expression vector may be YEp13, YCp50,pRs or pYEX vector. A promoter may be, for example, GAL or AOD promoter.A recombinant DNA may be introduced into an yeast by, for example,electroporation (Methods Enzymol., 194, 182-187 (1990)), a spheroplastmethod (Proc. Natl. Acad. Sci. USA, 84, 1929-1933 (1978)) and a lithiumacetate method (J. Bacteriol., 153, 163-168 (1983)).

A recombinant vector may further have a fragment for expressionalregulation which has a variety of functions for suppression,amplification or triggering of expression; a marker for selection of atransformant; a resistance gene to an antibiotic; or a gene encoding asignal for extracellular secretion.

A PHA synthase of the present invention may be prepared by culturing atransformant prepared by transforming a host with a recombinant vectorhaving a gene encoding the synthase to produce and accumulate a PHAsynthase as a gene product in the culture (cultured bacterium or culturesupernatant) and isolating the PHA synthase from the culture.

The transformant of the present invention may be cultured by a commonprocess used for culturing a host.

Culturing may be conducted by any of common microorganism culturingprocesses such as batch, flow batch, continuous culturing and reactorstyles.

For a transformant obtained using a bacterium such as E. coli as a host,a medium used for culturing may be a complete medium or synthetic mediumsuch as LB medium and M9 medium. A microorganism may be grown byaerobically culturing it at a culturing temperature of 25 to 37° C. for8 to 72 hours to accumulate a PHA synthase in bacterial cells, and theenzyme may be collected. Microbial growth requires a carbon sourceincluding sugars such as glucose, fructose, sucrose, maltose, galactoseand starches; lower alcohols such as ethanol, propanol and butanol;polyalcohols such as glycerol; organic acids such as acetic acid, citricacid, succinic acid, tartaric acid, lactic acid and gluconic acid; andaliphatic acids such as propionic acid, butanoic acid, pentanoic acid,hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoicacid, undecanoic acid and dodecanoic acid.

Examples of a nitrogen source include ammonia; ammonium salts such asammonium chloride, ammonium sulfate and ammonium phosphate; and naturalproduct derivatives such as peptone, meat extract, yeast extract, maltextract, casein decomposition products and corn steep liquor. Examplesof an inorganic material include potassium dihydrogen phosphate,potassium monohydrogen phosphate, magnesium phosphate, magnesium sulfateand sodium chloride. The culture medium may contain an antibiotic suchas kanamycin, ampicillin, tetracyclin, chloramphenicol and streptomycin.

When culturing a microorganism transformed using an expression vectorhaving an inducible promoter, a proper inducer suitable to the type ofthe promoter may be added to a culture medium. For example, the inducermay be isopropyl-β-D-thiogalactopyranoside (IPTG), tetracyclin orindoleacrylic acid (IAA).

A PHA synthase may be separated and purified by centrifuging andcollecting cells or a supernatant from a culture obtained and processingit by a technique such as cell disruption extraction, affinitychromatography, cation or anion exchange chromatography and gelfiltration alone or in combination as appropriate. Whether a purifiedmaterial is a desired enzyme may be determined by a usual method such asSDS polyacrylamide gel electrophoresis and Western blotting.

The present invention is not limited to the procedures as describedabove for culturing of a transformant using microorganism as a host,production of a PHA synthase by the transformant and accumulating it inmicroorganisms, and collection of the PHA synthase from the cells.

When culturing a transformant using a microorganism as a host for PHAproduction, the procedure may also be used in which the transformant iscultured using an appropriate medium composition and culturingconditions depending on factors such as the host used and theconstitution of a recombinant vector introduced in the host and the PHAis obtained from the culture. A medium or culturing conditions may bethe same as those illustrated for the above preparation of a PHAsynthase.

A PHA may be collected from cells most conveniently by extraction withan organic solvent such as chloroform as usual, but in an environmentwhere using an organic solvent such as chloroform is undesirable, theculture way be treated by a surfactant such as SDS, an enzyme such aslysozyme, or an agent such as EDTA, sodium hypochlorite and ammonia toremove bacterium components other than the PHA for collecting the PHA.

The present invention is not limited to the above procedures forculturing of a transformant using a microorganism as a host, productionof a PHA by and accumulation thereof in the transformant, and collectionof the PHA from the cells.

EXAMPLES

The present invention will be more specifically described with referenceto Examples although these Examples do not limit the technical range ofthis invention.

Example 1 Cloning of a PHA Synthase Gene of P91 Strain

P91 strain was cultured in 100 mL of LB medium (1% polypeptone, 0.5%yeast extract, 0.5% sodium chloride, pH 7.4) at 30° C. overnight andthen a chromosome DNA was separated and collected as described byMarmer. The chromosome DNA obtained was completely digested using arestriction enzyme BglII. A vector pUC18 was cleaned with a restrictionenzyme BamHI. After dephosphorylation of the terminals (MolecularCloning, Vol. 1, p. 572 (1989), Cold Spring Harbor Laboratory), thedigested vector and the chromosome DNA fragment after BglII completedigestion were ligated using a DNA ligation kit Ver. II (Takara ShuzoCo., Ltd.). The ligated DNA fragment was used to transform Escherichiacoli HB101 strain for preparing a chromosome DNA library for P91 strain.

Then, in order to select a DNA fragment comprising a PHA synthase geneof P91 strain, a probe was prepared. An oligonucleotide consisting ofthe sequences of SEQ ID NOs.:5 and 6 (Amersham-Pharmacia Biotech) wasprepared and used as a primer for PCR. An amplified fragment was used asa probe. Labeling of the probe was conducted using AlkPhosDirect(Amersham-Pharmacia Biotech). The probe thus obtained was used to selectan E. coli strain containing a recombinant plasmid comprising the PHAsynthase gene from the chromosome DNA library of P91 strain by colonyhybridization. From the selected strain, the plasmid was collected by analkali method to prepare a DNA fragment comprising a PHA synthase gene.

The gene fragment thus obtained was recombined in a vector pBBR122 (MoBi Tec) comprising a wide host range of replication region which did notbelong to IncP, IncQ or IncW in an incompatible group. The recombinantplasmid was transformed in Pseudomonas putida P91ml strain (a straindepleted of PHA synthesizing ability) by electroporation, and then theP91ml strain regained PHA synthesizing ability and exhibitedcomplementarity.

The fragment comprising a PHA synthase gene was sequenced by Sanger'ssequencing method. It was thus found that the fragment comprised a PHAsynthase gene having the sequences of SEQ ID NOs.: 2 and 4. SEQ ID NOs.:1 and 3 show the amino acid sequences coded by SEQ ID NOs.: 2 and 4,respectively.

Example 2 Recombination of a PHA Synthase Gene of P91 Strain to anExpression Vector

An oligonucleotide having a sequence around the initiation codon of thePHA synthase gene of SEQ ID NO.:2 (SEQ ID NO.:7) and an oligonucleotidehaving a sequence around the termination codon (SEQ ID NO.:8) weredesigned and synthesized (Amersham-Pharmacia Biotech). Theoligonucleotides were used as a primer for PCR to amplify the wholelength of the PHA synthase gene (LA-PCR kit; Takara Shuzo Co., Ltd.).

An oligonucleotide having a sequence around the initiation codon of thePHA synthase gene of SEQ ID NO.:4 (SEQ ID NO.:9) and an oligonucleotidehaving a sequence around the termination codon (SEQ ID NO.:10) weredesigned and synthesized (Amersham-Pharmacia Biotech). Theoligonucleotides were used as a primer for PCR to amplify the wholelength of the PHA synthase gene (LA-PCR kit; Takara Shuzo Co., Ltd.).

Each of the obtained PCR amplified fragments was completely digestedusing a restriction enzyme HindIII, and ligated to an expression vectorpTrc99A which had been truncated with a restriction enzyme HindIII anddephosphorylated (Molecular Cloning, Vol. 1, p. 5.7.2 (1989), ColdSpring Harbor Laboratory), using a DNA ligation kit Ver. II (TakaraShuzo Co., Ltd.).

Using the recombinant plasmids, Escherichia coli HB101 was transformedby a calcium chloride method (Takara Shuzo Co., Ltd.), and recombinantplasmids collected from the transformants were designated as pP91-C1(derived from SEQ ID NO.:2) and pP91-C2 (derived from SEQ ID NO.:4),respectively.

Example 3 PHA Production (1) Using a PHA Synthase Gene Recombinant E.coli

Using the recombinant plasmids obtained in Example 2, pP91-C1 (derivedfrom SEQ ID NO.:2) and pP91-C2 (derived from SEQ ID NO.:4), anEscherichia coli HB101fB (fadB deficient strain) was transformed by acalcium chloride method to prepare recombinant E. coli strains derivedfrom the recombinant plasmids, respectively.

Each of the pP91-C1 and pP91-C2 recombinant strains was inoculated to200 mL of M9 medium containing 0.5% yeast extract and 0.1% FPVA, and themedium was shaken at 37° C. with a rate of 125 strokes/min. After 24hours, the cells were collected by centrifugation, washed once with coldmethanol and lyophilized.

The lyophilized pellet was suspended in 100 mL of chloroform and thesuspension was stirred at 60° C. for 20 hours to extract a PHA. Afterfiltering the extract through a membrane filter with a pore size of 0.45μm, the filtrate was concentrated by rotary evaporation. Then, theconcentrate was re-suspended in cold methanol and the precipitant wascollected and dried in vacuo to provide a PHA. The PHA thus obtained wassubject to methanolysis as usual and analyzed using a gaschromatography-mass spectrometry apparatus (GC-MS, Shimazu QP-5050, EItechnique) to identify methyl-esterified PHA monomer units. The resultsare shown in Table 1.

TABLE 1 pP91-C1 pP91-C2 recombinant recombinant strain strain Cell dryweight 810 mg/L 800 mg/L Polymer dry weight  24 mg/L  23 mg/L Polymerdry weight/Cell  3%  3% dry weight Monomer unit composition (area ratio)3-Hydroxybutyric acid  0%  0% 3-Hydroxyvaleric acid  0%  0%3-Hydroxyhexanoic acid  0%  0% 3-Hydroxyheptanoic acid  5%  3%3-Hydroxyoctanoic acid  4%  5% 3-Hydroxynonanoic acid  9% 11%3-Hydroxydecanoic acid 10% 12% 3-Hydroxy-5-(4-fluorophenyl) 72% 69%valeric acid

Example 4 PHA Production (2) Using a PHA Synthase Gene Recombinant E.coli

Each of the pP91-C1 and pP91-C2 recombinant strains was inoculated to200 mL of M9 medium containing 0.5% yeast extract and 0.2% PxBA, andthen cultured with shaking at 37° C. with a rate of 125 strokes/min.After 24 hours, the cells were collected by centrifugation, washed oncewith cold methanol and lyophilized.

The lyophilized pellet was suspended in 100 mL of chloroform and thesuspension was stirred at 60° C. for 20 hours to extract a PHA. Afterfiltering the extract through a membrane filter with a pore size of 0.45μm, the filtrate was concentrated by rotary evaporation. Then, theconcentrate was re-suspended in cold methanol and only the precipitantwas collected and dried in vacuo to provide a PHA. The PHA thus obtainedwas subject to methanolysis as usual and analyzed using a gaschromatography-mass spectrometry apparatus (GC-Ms, Shimazu QP-5050, EItechnique) to identify methyl-esterified PHA monomer units. The resultsare shown in Table 2.

TABLE 2 pP91-C1 pP91-C2 recombinant recombinant strain strain Cell dryweight 750 mg/L 720 mg/L Polymer dry weight  4 mg/L  4 mg/L Polymer dryweight/Cell 0.5%  0.6%  dry weight Monomer unit composition (area ratio)3-Hydroxybutyric acid  0%  0% 3-Hydroxyvaleric acid  0%  0%3-Hydroxyhexanoic acid  0%  0% 3-Hydroxyheptanoic acid  2%  2%3-Hydroxyoctanoic acid  3%  3% 3-Hydroxynonanoic acid  5%  7%3-Hydroxydecanoic acid  5%  6% 3-Hydroxy-4-phenoxy-n- 85% 82% butyricacid

Example 5 Homology of a PHAsynthase Gene of P91 Strain

In the same manner as in Example 1, the chromosome DNA of P91 strain wasseparated and collected. Further, Pseudomonas olevorans ATCC29347,Pseudomonas putida Tk2440 and Pseudomonas aeruginose PA01 were culturedand the chromosome DNAs thereof were separated and collected in the sameas in Example 1.

Next, a probe for hybridization was prepared for confirming homology ofa PHAsynthase gene of P91 strain. In the same manner as in Example 2, anoligonucleotide having the sequences of SEQ ID NOs 7 and 8 weresynthesized. (Amersham-Pharmacia Biotech) PCR was conducted using thethus synthesized oligonucleotide as a primer and the chromosome DNA as atemplate. The obtained PCR amplified DNA fragments were used as a probe.Labeling of the probe using AlkphosDirect (Amersham-Pharmacia Biotech)was conducted to obtain the labeled probe referred to as “phaC1”. In thesame manner as above, an oligonucleotide having the sequences of SEQ IDNOs 9 and 10 were synthesized. (Amersham-Pharmacia Biotech) PCR wasconducted using the thus synthesized oligonucleotide as a primer and thechromosome DNA as a template. Labeling of the obtained PCR amplified DNAfragments was conducted in the same manner as above to obtain thelabeled probe referred to as “phaC2”.

Using the thus obtained probes, homology of the PHA synthase gene wasconfirmed by a dot-blot method. The thus prepared chromosome DNA wasalkalized and then blotted on a nylon film (Tropilon-45, produced byTropix Co.) by 1 μg at respective portions using a dot-blot apparatus(BRL).

The film was dried at 80° C. for two hours, then put in a vinyl bag. 3ml of the solution for hybridization prepared according to the recipe ofAlkPhosDirect was added thereto, and hybridization was conducted at 55°C. for one hour. 15 ng of the labeled probe phaC1 or phaC2 per 3 ml ofthe above hybridization solution (5 ng/ml) was added to the nylon film,and hybridization was conducted at 55° C. for 12 hours. And then thenylon film was put out from the vinyl bag, and washed two times for 10minutes at 55° C. with a first washing buffer according the recipe ofAlkiPhosDirect. And then after it was washed two times for 5 minutes atroom temperature with a second washing buffer according to the recipe ofAlkPhosDirect, a detecting step was carried out using CDP-Star attachedto AlkiPhosDirect according to the recipe.

Further, a detecting step was carried out under the same conditions asabove except that hybridization and first washing were conducted at 60°C. or 65° C. The results were shown in Table 3 and 4.

TABLE 3 Temperature (° C.) 55 60 65 Probe phaC1 Target DNA P91 Strain AA B ATCC29347 Strain C D D Probe phaC2 Target DNA P91 Strain A A BATCC29347 Strain C D D A: Strong Signal, B: Signal, C: Slight Signal, D:No Signal

TABLE 4 Temperature (° C.) 55 60 65 Probe phaC1 Target DNA P91 Strain AA B ATCC29347 Strain C D D KT2440 Strain B C D PAO1 Strain D D D ProbephaC2 Target DNA P91 Strain A A B ATCC29347 Strain C D D KT2440 Strain DC D PAO1 Strain D D D A: Strong Signal, B: Signal, C: Slight Signal, D:No Signal

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 10 <210> SEQ ID NO 1 <211> LENGTH: 559<212> TYPE: PRT <213> ORGANISM: Pseudomonas putida P91 <220> FEATURE:<223> OTHER INFORMATION: Polyhydroxyalkanoate synthase <400> SEQUENCE: 1Met Ser Asn Lys Asn Asn Asp Asp Leu Gln Ar #g Gln Ala Ser Glu Asn1               5    #                10   #                15Thr Leu Gly Leu Ser Pro Ile Ile Gly Leu Ar #g Arg Lys Asp Leu Leu            20       #            25       #            30Ser Ser Ala Arg Met Val Leu Arg Gln Ala Il #e Lys Gln Pro Leu His        35           #        40           #        45Ser Ala Lys His Val Ala His Phe Gly Leu Gl #n Leu Lys Asp Val Ile    50               #    55               #    60Phe Gly Lys Ser Gly Leu Gln Pro Glu Gly As #p Asp Arg Arg Phe Ser65                   #70                   #75                   #80Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Ar #g Arg Tyr Leu Gln Thr                85   #                90   #                95Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Tr #p Ile Gly Asn Ser Asn            100       #           105       #           110Leu Ser Glu Gln Asp Ile Ser Arg Ala His Ph #e Val Ile Asn Leu Met        115           #       120           #       125Thr Glu Ala Met Ala Pro Thr Asn Ser Ala Al #a Asn Pro Ala Ala Val    130               #   135               #   140Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Le #u Leu Asp Gly Leu Ser145                 1 #50                 1 #55                 1 #60His Leu Ala Lys Asp Met Val His Asn Gly Gl #y Met Pro Ser Gln Val                165   #               170   #               175Asn Met Asp Ala Phe Glu Val Gly Lys Asn Le #u Ala Thr Thr Glu Gly            180       #           185       #           190Ala Val Val Phe Arg Asn Asp Val Leu Glu Le #u Ile Gln Tyr Arg Pro        195           #       200           #       205Ile Thr Glu Gln Val His Glu Lys Pro Leu Le #u Val Val Pro Pro Gln    210               #   215               #   220Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pr #o Glu Lys Ser Leu Ala225                 2 #30                 2 #35                 2 #40Arg Phe Cys Leu Arg Ser Thr Val Gln Thr Ph #e Ile Val Ser Trp Arg                245   #               250   #               255Asn Pro Asn Lys Ser Gln Arg Glu Trp Gly Le #u Ser Thr Tyr Ile Asp            260       #           265       #           270Ala Leu Lys Glu Ala Val Asp Val Val Leu Al #a Ile Thr Gly Ser Lys        275           #       280           #       285Asp Leu Asn Met Leu Gly Ala Cys Ser Gly Gl #y Ile Thr Cys Thr Ala    290               #   295               #   300Leu Val Gly His Tyr Ala Ala Leu Gly Glu Ly #s Lys Val Asn Ala Leu305                 3 #10                 3 #15                 3 #20Thr Leu Leu Val Ser Val Leu Asp Thr Thr Le #u Asp Thr Gln Val Ala                325   #               330   #               335Leu Phe Val Asp Glu Gln Thr Leu Glu Ser Al #a Lys Arg His Ser Tyr            340       #           345       #           350Gln Ala Gly Val Leu Glu Gly Arg Asp Met Al #a Lys Val Phe Ala Trp        355           #       360           #       365Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Tr #p Val Asn Asn Tyr Leu    370               #   375               #   380Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Le #u Phe Trp Asn Asn Asp385                 3 #90                 3 #95                 4 #00Ile Thr Arg Leu Pro Ala Ala Phe His Gly As #p Leu Ile Glu Met Phe                405   #               410   #               415Lys Asn Asn Pro Leu Val Arg Pro Gly Ala Le #u Glu Val Cys Gly Thr            420       #           425       #           430Pro Ile Asp Leu Ser Gln Val Thr Thr Asp Il #e Phe Ser Val Ala Gly        435           #       440           #       445Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cy #s Tyr Lys Ser Ala Gln    450               #   455               #   460Leu Phe Gly Gly Lys Val Glu Phe Leu Leu Se #r Asn Ser Gly His Ile465                 4 #70                 4 #75                 4 #80Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Ly #s Ser Arg Tyr Met Thr                485   #               490   #               495Ser Ser Glu Met Pro Ala Gln Ala Asp Asp Tr #p Gln Glu Asn Ser Thr            500       #           505       #           510Lys His Thr Asp Ser Trp Trp Leu Tyr Trp Gl #n Ala Trp Leu Ala Glu        515           #       520           #       525Arg Ser Gly Ala Leu Lys Pro Ala Pro Ala Ly #s Leu Gly Asn Lys Ala    530               #   535               #   540Tyr Pro Ser Ala Glu Ala Ser Pro Gly Thr Ty #r Val His Glu Arg545                 5 #50                 5 #55 <210> SEQ ID NO 2<211> LENGTH: 1680 <212> TYPE: DNA<213> ORGANISM: Pseudomonas putida P91 <220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)...(1680) Polyhydroxyalkanoate syntha #se encoding      sequence <400> SEQUENCE: 2atgagtaaca agaacaacga tgacctgcag cgccaagcct ctgaaaacac cc#tgggcctg     60agccccatca ttggcctgcg ccgaaaggat ttgctgtctt cggcccggat gg#tgctgcgt    120caggccatca agcaaccgct gcacagtgcc aagcacgtcg cgcatttcgg cc#tgcagctc    180aaggacgtga tcttcggcaa gtccggcctg cagccggagg gcgacgaccg cc#gcttcagc    240gacccggcct ggagccagaa cccgctgtac cgccgctacc tgcagaccta cc#tggcctgg    300cgcaaggaac tgcacgactg gatcggcaac agcaacctgt cggagcagga ca#tcagccgc    360gcgcacttcg tcatcaacct gatgaccgag gccatggccc ccaccaacag cg#cggccaac    420ccggcagcgg tcaagcgctt cttcgaaacc ggtggcaaga gcctgctcga cg#gcctgtcg    480cacctggcca aggacatggt ccacaacggc ggcatgccca gccaggtcaa ca#tggacgcc    540ttcgaggtgg gcaagaacct ggccaccacc gagggcgccg tggtatttcg ca#acgacgtg    600ctggagctga tccagtaccg cccgatcacc gagcaggtgc acgaaaagcc gc#tgctggtg    660gtaccgccgc agatcaacaa gttctacgtc ttcgacctca gcccggaaaa ga#gcctggcg    720cgcttctgcc tgcgctccac ggtgcagacc ttcatcgtga gctggcgcaa cc#ccaacaag    780tcccagcgcg agtggggcct gtcgacctac atcgatgcgc tcaaggaggc cg#tcgacgtg    840gtgctggcaa tcaccggcag caaggacctg aacatgctcg gtgcctgctc cg#gcggcatc    900acctgcaccg cgctggtggg ccactacgcg gcactgggcg agaagaaggt ca#atgccctg    960accctgctgg tgagcgtgct cgacaccacc ctcgacaccc aggtggcgct gt#tcgtcgac   1020gagcagaccc tggagtcggc caagcgccat tcctaccagg ccggtgtgct cg#aaggccgc   1080gacatggcca aggtgttcgc ctggatgcgc cccaacgacc tgatctggaa ct#actgggtc   1140aacaactacc tgctcggcaa cgagccgccg gtgttcgaca tcctgttctg ga#acaacgac   1200atcacgcgcc tgcccgccgc cttccacggc gacctgatcg aaatgttcaa ga#acaacccg   1260ctggtgcgtc ccggtgcact ggaagtgtgc ggcacgccga tcgacctgag cc#aggtcacc   1320accgacatct tcagcgtggc cggcaccaac gatcacatca ccccatggaa gt#cctgctac   1380aagtcggcgc agctgttcgg cggcaaggtc gagttcctgc tgtccaacag cg#ggcatatc   1440cagagcatcc tcaacccgcc gggcaacccc aagtcgcgct acatgaccag ca#gcgagatg   1500ccggcccagg ccgacgactg gcaggagaac tccaccaagc acaccgattc ct#ggtggctg   1560tactggcagg cgtggctggc cgagcgctcc ggcgcactca agccggcacc cg#ccaagctg   1620ggcaacaagg cctacccgag cgccgaagcg tcgcccggca cctacgtcca cg#aacgctga   1680 <210> SEQ ID NO 3 <211> LENGTH: 560 <212> TYPE: PRT<213> ORGANISM: Pseudomonas putida P91 <220> FEATURE:<223> OTHER INFORMATION: Polyhydroxyalkanoate synthase <400> SEQUENCE: 3Met Lys Asp Lys Pro Ala Lys Pro Gly Val Pr #o Thr Pro Ala Ala Tyr1               5    #                10   #                15Leu Asn Val Arg Ser Ala Ile Ser Gly Leu Ar #g Gly Arg Asp Leu Leu            20       #            25       #            30Ser Thr Val His Gln Leu Gly Arg His Gly Le #u Arg His Pro Leu His        35           #        40           #        45Thr Ala Arg His Leu Leu Ala Leu Gly Gly Gl #n Leu Gly Arg Val Met    50               #    55               #    60Leu Gly Asp Thr Pro Tyr Gln Pro Ser Pro Ar #g Asp Thr Arg Phe Asn65                   #70                   #75                   #80Asp Pro Ala Trp Gln Leu Asn Pro Leu Tyr Ar #g Arg Gly Leu Gln Ala                85   #                90   #                95Tyr Leu Ala Trp Gln Gln Gln Thr Cys Gln Tr #p Ile Asp Glu Ser Gln            100       #           105       #           110Leu Asp Asp Asp Asp Arg Ala Arg Ala His Ph #e Val Phe Ser Leu Leu        115           #       120           #       125Asn Asp Ala Met Ser Pro Ser Asn Thr Leu Le #u Asn Pro Ala Ala Val    130               #   135               #   140Lys Glu Leu Leu Asn Ser Gly Gly Leu Ser Le #u Val Arg Gly Leu Asn145                 1 #50                 1 #55                 1 #60His Leu Leu Asp Asp Leu Arg His Asn Asp Gl #y Leu Pro Arg Gln Val                165   #               170   #               175Asn Pro Asp Ala Phe Glu Val Gly Arg Asn Le #u Ala Ser Thr Ala Gly            180       #           185       #           190Ala Val Val Phe Arg Asn Glu Leu Leu Glu Le #u Ile Gln Tyr Arg Pro        195           #       200           #       205Met Ser Glu Lys Gln Tyr Ala Arg Pro Leu Le #u Val Val Pro Pro Gln    210               #   215               #   220Ile Asn Lys Phe Tyr Ile Phe Asp Leu Ser Pr #o Thr Asn Ser Phe Val225                 2 #30                 2 #35                 2 #40Gln Tyr Ala Leu Lys Asn Gly Leu Gln Thr Ph #e Met Ile Ser Trp Arg                245   #               250   #               255Asn Pro Asp Ala Arg His Arg Glu Trp Gly Le #u Ser Ser Tyr Val Ala            260       #           265       #           270Ala Val Glu Glu Ala Met Asn Val Cys Arg Se #r Ile Thr Gly Ser Arg        275           #       280           #       285Asp Val Asn Leu Leu Gly Ala Cys Ala Gly Gl #y Leu Thr Ile Ala Ala    290               #   295               #   300Leu Gln Gly His Leu Gln Ala Lys Arg Gln Me #t Arg Arg Val His Ser305                 3 #10                 3 #15                 3 #20Ala Thr Tyr Leu Val Ser Leu Leu Asp Ser Gl #n Phe Asp Ser Pro Ala                325   #               330   #               335Ser Leu Phe Ala Asp Glu Gln Thr Leu Glu Al #a Ala Lys Arg Arg Ser            340       #           345       #           350Tyr Gln Gln Gly Val Leu Glu Gly Arg Glu Me #t Ala Arg Val Phe Ala        355           #       360           #       365Trp Met Arg Pro Asn Asp Leu Ile Trp Asn Ty #r Phe Val Asn Asn Tyr    370               #   375               #   380Leu Leu Gly Lys Ala Pro Pro Ala Phe Asp Il #e Leu Tyr Trp Asn Asn385                 3 #90                 3 #95                 4 #00Asp Asn Ser Arg Leu Pro Ala Ala Leu His Gl #y Asp Leu Leu Asp Phe                405   #               410   #               415Phe Lys Phe Asn Pro Leu Thr His Ala Asp Gl #y Leu Glu Val Cys Gly            420       #           425       #           430Thr Pro Ile Asp Leu Asn Lys Val Thr Val As #p Ser Phe His Val Ala        435           #       440           #       445Gly Ser Asn Asp His Ile Thr Pro Trp Asp Al #a Val Tyr Arg Ser Ala    450               #   455               #   460Leu Leu Leu Gly Gly Glu Arg Arg Phe Val Le #u Ala Asn Ser Gly His465                 4 #70                 4 #75                 4 #80Val Gln Ser Ile Leu Asn Pro Pro Gly His Pr #o Lys Ala His Phe Val                485   #               490   #               495Glu Asn Pro Arg Leu Ser Ser Asp Pro Arg Al #a Trp Tyr His Asp Ala            500       #           505       #           510Gln Lys Val Glu Gly Ser Trp Trp Pro Gln Tr #p Leu Asp Trp Ile Gln        515           #       520           #       525Ala Arg Ser Gly Ala Gln Arg Glu Thr Arg Le #u Ser Leu Gly Ser Ala    530               #   535               #   540Asn Tyr Pro Pro Met Asp Pro Ala Pro Gly Th #r Tyr Val Leu Val Arg545                 5 #50                 5 #55                 5 #60<210> SEQ ID NO 4 <211> LENGTH: 1683 <212> TYPE: DNA<213> ORGANISM: Pseudomonas putida P91 <220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1)...(1683) Polyhydroxyalkanoate syntha #se encoding      sequence <400> SEQUENCE: 4atgaaagaca agcccgcgaa gcccggggta ccgacccccg ctgcctatct ca#acgtgcgc     60agcgccatca gtggcctgcg cggtcgcgac ctgctgtcga cggtgcacca gc#tggggcgc    120cacggcctgc gtcacccgct gcacacggcg cgccacctgc tggcgctggg tg#gccagctg    180gggcgcgtga tgctgggcga taccccctac cagccctcgc cacgcgacac cc#gcttcaac    240gacccggcct ggcagctcaa cccgctgtac cgacgcggcc tgcaggccta cc#tggcctgg    300cagcagcaga cctgccagtg gatcgacgag agccagctgg acgacgatga cc#gcgcccgc    360gcgcacttcg tgttctcgct gctcaacgat gcaatgtcgc ccagcaacac cc#tgctcaac    420ccggcggcgg tcaaggagct gctgaactcc ggcgggctga gcctggtgcg cg#gcttgaac    480cacctgctcg acgacctgcg ccacaacgac ggcctgccac gccaggtcaa cc#cggacgcc    540ttcgaggtgg gcaggaacct ggccagcacc gccggcgcgg tggtgtttcg ca#acgagctg    600ctggagctga tccagtaccg cccgatgagc gaaaaacagt acgcccggcc cc#tgctggtg    660gtgccgccgc agatcaacaa gttctacatc ttcgacctca gcccgaccaa ca#gctttgtg    720cagtacgccc tcaagaacgg cctgcagacc ttcatgatca gctggcgcaa cc#ccgacgcc    780cggcatcgcg aatggggcct gtcgagctac gtggcggcgg tcgaggaagc ca#tgaacgtg    840tgccgctcga tcaccggcag ccgcgacgtc aacctgcttg gcgcctgtgc cg#gcgggttg    900accatcgcgg ccctgcaggg tcacctgcag gccaagcgcc agatgcgccg gg#tgcacagc    960gccacctacc tggtcagcct gctcgacagc cagttcgaca gccccgccag cc#tgttcgcc   1020gacgagcaga ccctggaggc ggccaagcgc cgctcctacc agcagggcgt gc#tggagggc   1080cgcgagatgg cacgggtgtt cgcctggatg cgccccaacg acctgatctg ga#actacttc   1140gtcaacaact acctgctggg caaggcgccc ccggcattcg acatcctgta ct#ggaacaac   1200gacaacagcc gcctgccggc cgcgctgcac ggcgatctgc tggacttctt ca#aattcaac   1260ccgctgacgc acgccgacgg cctcgaggta tgcggcacgc cgatcgacct ga#acaaggtc   1320acggtggaca gcttccacgt ggccggcagc aacgaccaca tcaccccgtg gg#acgcggtg   1380taccgctcgg ccctgctgct gggcggcgag cggcgcttcg tgctggccaa ca#gcgggcat   1440gtgcagagca tcctcaaccc accgggccac cccaaggcgc attttgtcga ga#accccagg   1500ctgagcagcg acccgcgggc ctggtaccac gatgcgcaga aggtcgaggg ca#gctggtgg   1560ccgcagtggc tcgactggat acaggcgcgc tccggtgcgc agcgcgaaac cc#gcctgtcg   1620ctgggcagcg ccaattaccc tcccatggac cccgcacccg gcacctacgt gc#tggtgcgc   1680 tga                   #                  #                   #           1683 <210> SEQ ID NO 5 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Probe sequence <400> SEQUENCE: 5tgctggaact gatccagtac             #                  #                   # 20 <210> SEQ ID NO 6 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Probe sequence <400> SEQUENCE: 6gggttgagga tgctctggat gtg            #                  #                23 <210> SEQ ID NO 7 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer sequence for PCR <400> SEQUENCE: 7cagccaagct tgtactcgtc tcaggacaac          #                  #           30 <210> SEQ ID NO 8 <211> LENGTH: 29 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer sequence for PCR <400> SEQUENCE: 8agagataagc ttgcggcatg cgcgagccc          #                  #            29 <210> SEQ ID NO 9 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer sequence for PCR <400> SEQUENCE: 9cattgaagct ttggttgatg gcctgacgac          #                  #           30 <210> SEQ ID NO 10 <211> LENGTH: 29 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer sequence for PCR <400> SEQUENCE: 10ctccaagctt cggtcgcggg tcttcatcc          #                  #            29

What is claimed is:
 1. An isolated polyhydroxyalkanoate synthase havingthe amino acid sequence of SEQ ID NO:
 1. 2. An isolatedpolyhydroxyalkanoate synthase having an amino acid sequence, which is atleast 95% homologous with the amino acid sequence of SEQ ID NO:
 1. 3. Amethod for preparing the polyhydroxyalkanoate synthase according toclaim 1 or 2, comprising the steps of: culturing the transformedmicroorganism harboring a recombinant vector containing a DNA sequenceencoding the polyhydroxyalkanoate synthase in a medium; expressing saidDNA; and isolating the polyhydroxyalkanoate synthase from the cultureobtained.
 4. A method according to claim 3, wherein the DNA has thesequence according to SEQ ID NO: 2.